CP-AMPAR upregulation is more robust in upscaled Ppt1^−/− neurons. A, Representative calcium imaging (GCaMP) video still frames of vehicle control or TTX-treated (1 μm, 48 h) WT and Ppt1^−/− cortical neurons. Scale bar = 20 µm. B, Representative ΔF/F₀ traces for ten synapses in each condition (WT, Ppt1^−/−, TTX-WT, and TTX-Ppt1^−/−). Open circles denote NASPM-insensitive synapses and closed circles denote NASPM-sensitive synapses. C, Quantification of the average synaptic calcium activity (number of frames >3 SDs from baseline) for all cells from each condition before NASPM infusion (i.e., at baseline). Two-way ANOVA: interaction genotype × TTX-treatment F_(1,16) = 6.349, *p = 0.0228; main effect of genotype = F_(1,16) = 3.205, p = 0.1669; main effect of treatment F_(1,16) = 37.15, ****p < 0.0001. Tukey's multiple comparison indicated on graph: *p = 0.0442 WT Veh versus WT TTX; ****p < 0.0001 Ppt1^−/− Veh versus Ppt1^−/− TTX; *p = 0.0304 WT TTX versus Ppt1^−/− TTX. n = 4–6 cells/group, cells taken from at least 2 independent cultures. D, Quantification of the average synaptic calcium activity (number of frames >3 SDs from baseline) for all synapses in all cells from each condition before, during, and following NASPM (1 μm) bath infusion. Repeated measures two-way ANOVA: interaction genotype/TTX × NASPM F_(3,16) = 15.74, ****p < 0.0001; main effect of genotype/TTX F_(3,16) = 1.731, p = 0.2009; main effect of NASPM F_(1,16) = 133.4, ****p < 0.0001. Bonferroni's multiple comparison test shown on graph for baseline, NASPM: **p = 0.0067 WT Veh; ****p < 0.0001 WT TTX; ****p < 0.0001 Ppt1^−/− TTX. n = 4–6 cells/group, cells taken from at least 2 independent cultures. E, Quantification of the proportion of NASPM-sensitive synapses in WT, TTX-treated WT, Ppt1^−/−, and TTX-treated Ppt1^−/− cortical neurons expressed as the percentage of synapses inhibited during NASPM infusion. Two-way ANOVA: interaction genotype × TTX F_(1,16) = 6.167, *p = 0.0245; main effect of genotype F_(1,16) = 3.126, p = 0.0961; main effect of treatment F_(1,16) = 82.29, ****p < 0.0001. n = 4–6 cells/group, cells taken from at least 2 independent cultures. Data represent mean ± SEM.